Project Summary We know very little regarding SULT allostery. The few examples that exist tell us that their binding is physiologically relevant, SULT isoform specific, and that they can function as activators, inhibitors and specificity modulators. Their binding pockets are, at least in certain cases, capable of binding complex classes of compounds - as if they evolved to provide general sensing mechanisms. There are no structures of SULT-bound allosteres. This proposal stands to radically advance our understanding of SULT allostery and, in so doing, sulfuryl-transfer biology. Aim I. A novel spin-label NMR method was designed specifically to determine SULT allostere-binding sites. The method was used to obtain the structure of the first SULT- bound allostere, EGCG (preliminary data). The structure reveals where EGCG binds, why it inhibits the enzyme, and the molecular secrets of its high isoform specificity, which we will use to design allosteres for other isoforms. Six such structures across four SULT isoforms (1A1, 1A3, 1E1 and 2A1) will be determined. The allosteres and SULTs were selected based on biomedical relevance, unique mechanistic characteristics, and broad targeting of important metabolic areas: drugs and xenobiotics, catecholamine neurotransmitters, estrogens, and androgens. Aim II. Screens with each of the four isoforms will be used to isolate new allosteres from: small-molecule libraries prepared from human tissue; microbiota, toxin and bioactive-compound libraries; and in-silico human-metabolite libraries. Limited preliminary studies yielded eight new putative allosteres; four were tested, all were high-affinity allosteres, three were endogenous human metabolites; among them was tetrahydrobiopterin (THB), an essential cofactor in dopamine biosynthesis. THB proved to be a potent allosteric inhibitor of SULT1A3 (which inactivates dopamine) and may be involved in feedback regulation of dopamine activity. We anticipate many new allosteres will be discovered and that these linkages will begin to reveal the true depth and complexity of SULT metabolic networks. Aim III. There are many situations in which the ability to activate, inhibit or otherwise modulate the catalytic function of a specific SULT isoform is expected to be beneficial in managing disease. We are currently not well informed enough to design and build such effectors. The structures obtained from Aim I will change this situation. Each structure provides a new target against which isoform-specific allosteres can be designed, synthesized and tested, and we will use them to do so. Using structures, all atom molecular dynamics, in-silico synthesis, and protein-function studies, templates for the synthesis of small-molecule libraries will be constructed. Templates will be created from simplified molecular scaffolds with known allosteric properties, and will indicate where R-groups with particular physicochemical properties should be inserted to achieve isozyme specificity. These efforts will produce unique and powerful probes that may open new therapeutic avenues, and can be used to explore SULT function in unprecedented ways.